BREADFRUIT EXTRACT AS SKIN LIGHTENER 120 �--------------------------------. 'ii 100 � ! 8 C 'E la 'i :E 80 60 40 � 20 0 Control 10 ug/rrt. Kojic acid 10 ug/rnLA lnclsus Sample * 4.5 ug/rrt. Artocarpln 10 ug/ml. Hydroqulnone 51 Figure 6. Effects of A. incisus ether extract, hydroquinone, kojic acid, and artocarpin on melanin production of melanocyte Bl6Fl melanoma cells. Data are expressed as percent of control, and each column represents mean ± S.D. of triplicate study. Significantly different from the control value: *p 0.5, **p 0.01. Generally, skin pigmentation is determined by melanin synthesis within melanosomes and their distribution to keratinocytes within the epidermal melanin unit. Focusing on melanin synthesis, the rate-limiting step is the hydroxylation of tyrosine to DOPA by the enzyme tyrosinase. Since melanin production requires tyrosine, some mechanism, such as transportation of this amino acid through the melanosomal membrane, may be also a critical factor in limiting pigment production (12,13). As we know, kojic acid, a g-pyrone derivative, can strongly inhibit tyrosinase activity by chelation of the copper ion(s) at the active center of the enzyme (14, 15 ). For artocarpin, as mentioned above, its tyrosinase-inhibitory activity could not be observed. This probably results from its steric hindrance structure since a 4-substituted resorcinol skeleton, coupled with a C3 sub­ stituent, is present in the flavone structure of artocarpin (16). Therefore, the melano­ genesis-inhibitory activity of artocarpin may mainly involve the transportation of tyro­ sine through the melanosomal membrane, a subject that we need to clarify in the future. CELL VIABILITY As an attempt to find out if the reduction of melanin content in B16Fl cell treated with the ether extract causes the reduction of the viable cells, the determination of the effect of such an extract on cell proliferation was performed. B16Fl cells were treated with various concentrations (10, 25, and 100 µg/ml) of A. incisus ether extract for three days, and the number of cells was then determined. The numbers of viable cells were evaluated by staining cells with blue dye. As shown in Figure 7, A. incisus extract induced the dose-dependent inhibition of cell growth in B 16Fl cells. The inhibitory effects on B16Fl cells were less profound at a low dose (25 µg/ml) of the extract, whereas significant arrest of cell growth was observed at a concentration of 100 µg/ml. High doses of the extract may cause cell apoptosis and/or alteration of cell attachment, which is an important characteristic for the proliferation of adherent cells. The effect of kojic acid, A. incisus extract, artocarpin, and hydroquinone on the prolif­ eration of melanocyte B 16F 1 cells is shown in Figure 8. The obtained results indicate

52 JOURNAL OF COSMETIC SCIENCE 30.00 "I""" 25.00 m 20.00 C "jjj 15.00 � u 10.00 C m 5.00 ::: 0.00 0 2 3 4 5 Time (day) --+- Control - 10 ug/ml A incius extract -6- 25 ug/ml A incius extract -x- 100 ug/ml A incius extract Figure 7. Effect of A. incisus ether extract on viability of melanocyte B16Fl melanoma cells. Each point represents mean ± S.D. of triplicate study. a • 120 100 80 60 40 20 0 Control * 10 ug/ml Kojic acid * 10 ug/mLA. incisus Sample 4.5 ug/ml Artocarpln ** 10 ug/ml Hydroqulnona Fi gur e 8. Effects of A. incisus ether extract, hydroquinone, kojic acid, and artocarpin on viability of melanocyte B16Fl melanoma cells. Data are expressed as percent of control, and each column represents mean ± S.D. of triplicate study. Significantly different from the control value: *p 0.5, **p 0.01. that the percent viability of cells after being treated with kojic acid, A. incisus extract, artocarpin, and hydroquinone for three days was 90.02%, 91.39%, 79.17%, and 79.26%, respectively, as compared to the control cells. The microscopic technique was also used to investigate the phenotypic appearance of melanocyte cells to confirm the effect of these agents on melanocyte morphology. As shown in Figures 8 and 9, the number of cells markedly decreased after being treated with hydroquinone. Cell damage may result in a decrease in the production of melanin. Additionally, the dendrite